supporting information - pnas · 2014. 10. 5. · supporting information fu et al....

Supporting InformationFu et al. 10.1073/pnas.1408836111SI Materials and MethodsCloning, Expression, and Purification. Full-length human RRS(residues 1–660) and AIMP1 (residues 1–312) genes were in-serted simultaneously into pETDuet vector. Full-length humanQRS (residues 1–775) was inserted into the pET28a vector. BothQRS and RRS contained a His6 tag at the N terminus. Theplasmids containing the RRS gene, the AIMP1 gene, and theQRS gene were cotransformed into E. coli Rosetta (DE3), andthe bacteria were grown in LB broth medium. Initially, theprotein complexes were purified by an Ni2+ column using animidazole gradient in 25 mMTris·HCl (pH7.5), 300 mMNaCl, 5mM 2-mercaptoethanol, and 5% glycerol. The eluted proteincomplex was purified further with an ion-exchange column(Mono-Q) followed by gel filtration on a Superdex 200 columnequilibrated with 25 mM Tris·HCl (pH7.5), 150 mM NaCl, 5mM DTT, and 5% glycerol. The RQA1 complex used for crys-tallization was concentrated to 15 mg/mL. To analyze the sub-complex formation by gel-filtration chromatography, full-lengthhuman KRS (residues 1–597) and AIMP2 (residues 1–320) werecloned into pET28a. Full-length human AIMP1 (residues 1–312)and RRS (residues 1–660) also were inserted into pCDF andpET21a, respectively, for biochemical analyses.

Crystallization and Data Collection. Crystals of the RQA1 complexwere grown at 18 °C using the hanging-drop vapor-diffusionmethod from 5–10% methyl-pentene-diol, 0.1 M Tris·HCl (pH7.4), 0.2 M NaCl. Diffraction data were collected at −170 °Cusing crystals flash-frozen in crystallization buffer containing30% (wt/vol) glycerol. The crystals belong to space group C2221with dimensions of a = 102.7 Å, b = 313.3 Å, and c = 161.8 Å andcontained one complex in the asymmetric unit. Diffraction datafrom native crystals were collected at 0.9795 Å on beamline 5Cat the Pohang Advanced Light Source (Pohang, South Korea)and Photon Factory (Japan). The datasets were processed usingthe HKL2000 program (1).

Gel-Filtration Analyses. To determine whether AIMP1 or RRSsform the ternary complex, size-exclusion chromatography experi-ments for AIMP1 (M1–M4) and RRS (R1 or R2) mutants wereperformed in the presence of wild-type counterparts using a Su-perdex 200 16/60 column (GE Healthcare) at 4 °C. Runningbuffer contained 25 mM Tris·HCl (pH 7.5), 150 mM NaCl, 5 mMDTT, and 5% glycerol. The three (RRS, QRS, and AIMP1) orfive (RRS, QRS, KRS, AIMP1, and AIMP2) components of MSCwere mixed in an equimolar ratio and were incubated in ice for 1 hbefore being injected into the column. Interactions betweenAIMP2 (residues 40–80), AIMP1 (residues 1–73), and His-RRSN-terminal helix (residues 1–74) were analyzed by a Superdex 7510/300 column (GE Healthcare) at 4 °C.

GST Pull-Down Assay. Interactions between AIMP2 (residues 40–100) and the wild-type (or mutant) RQA1 complex were exam-ined by GST-mediated pull-down assays. GST-AIMP2 (residues40–100) was incubated with the ternary RQA1 complex containingwild-type or mutant AIMP1 in the presence of Glutathione Se-pharose resin at 4 °C for 2 h. After extensive washing with PBS,the complex was eluted with 10 mM reduced glutathione.

SAXS. SAXS analyses of the human RQA1 complex (1, 2, 3, and 5mg/mL) or the human RQA1–KA2 complex (1, 2, 3, and 5 mg/mL)were performed using the 4CSAXS II beamline of the PohangLightSource II with 3GeVpower at the PohangUniversity of Science and

Technology (Korea). All sample solutions were prepared in thebuffer [25 mM Tris (pH 7.5), 150 mM NaCl, 5 mM DTT, 5%glycerol]. The light source from the in-vacuum undulator 20 (IVU20: 1.4-m length, 20-mm period) of the Pohang Light Source IIstorage ring was focused using a vertical focusing toroidal mirrorcoated with rhodium andmonochromatized with an Si (111) doublecrystal monochromator, yielding an X-ray beam wavelength of0.675 Å (18.360 keV).The X-ray beam size at the sample stage was 0.2 (V) × 0.6

(H) mm2. A 2D charge-coupled detector (SX165, Mar USA, Inc.)was used with a sample-to-detector distance of 2.00 m. Themagnitude of scattering vectors [q = (4π/λ) sin θ; where 2θ is thescattering angle and λ is the wavelength of the X-ray beam source]was 0.10 nm−1 < q < 3.36 nm−1. The scattering angle was cali-brated with a polyethylene-b-polybutadiene-b-polystyrene blockcopolymer standard. Solution sample cells with 10-μm-thick micawindows, a volume of 50 μL, and an X-ray beam path length of0.8 mm were used. To monitor radiation damage, the SAXS datawere collected in five successive frames of 0.1 min each. All scat-tering measurements were carried out at 4 °C using an FP50-HLrefrigerated circulator (JULABO).Each 2D SAXS pattern was averaged circularly from the beam

center and normalized to the transmitted X-ray beam intensity,which was monitored with a scintillation counter placed behindthe sample. The scattering of the buffer solution was used as theexperimental background. The theoretical SAXS curves werecalculated from the crystal structures using the CRYSOL pro-gram (2).

Aminoacylation Assay.The arginylationor glutaminylation assaywascarried out in a buffer containing 20 mM Hepes (pH 7.5), 20 mMKCl, 10 mM MgCl2, 4 mM DTT, 5 mM ATP, tRNA (yeast totaltRNA, 4 mg/mL), and [3H] arginine or glutamine (60 Ci/mmol). Toobtain kinetic parameters (km, kcat, and kcat/km), human tRNAArg

(CCG), and human tRNAGln (CUG) were synthesized by in vitrotranscription and used with concentrations from 0.2–100 mM.Reactions were initiated by the addition of various RQA1 com-plexes or free RRS (or QRS) (40 nM) at 37 °C. Aliquots (15 uL)were taken at different time points and quenched on Whatmanfilter pads (catalog no. 1003323, grade 3, 2.3 cm) that were pre-soaked with 5% trichloroacetic acid (TCA). The pads were washedthree times for 10 min each with 5% cold TCA and with 100% coldethanol. The washed pads then were dried and placed into vials(Wheaton 986701) with 5mL of the mixture solution. Radioactivitywas quantified in a scintillation counter (Perkin-Elmer).

Western Blot. Purified GST (control), GST-RRS (Ha; residues1–30), and GST-AIMP1 (N-140) were mixed with FLAG-RRS(Hb; residues 38–68) at a 1:1 molar ratio and were incubated at4 °C for 3 h in the presence of glutathione resin. Beads bound tothe samples were washed extensively with PBS and eluted withPBS containing 10 mM glutathione. Western blotting of GST,GST–RRS (Ha), GST–AIMP1 (N-140) and FLAG–RRS (Hb)were performed with α-GST and α-FLAG antibodies, respectively,following the transfer of electrophoretically separated proteins toPVDF membrane using standard immunoblotting techniques.

Circular Dichroism Analysis. The secondary structure of the N-terminal domain of RRS (N-74; Ha and Hb) was monitored bycircular dichroism spectrophotometer (Jasco J-715) at wavelengthsof 190–250 nm at 18 °C. RRS (N-74; 100 uM) was prepared inbuffer containing 20 mM Tris-Cl (pH 7.5) and 100 mM NaCl.

Fu et al. www.pnas.org/cgi/content/short/1408836111 1 of 13

www.pnas.org/cgi/content/short/1408836111

Mutagenesis. All mutants used in this study were created by PCR-based methods. The mutant proteins were purified by affinitychromatography followed by anion exchange and gel-filtrationchromatography, as described above. For gel-filtration analyses, fourAIMP1mutants (M1,M2,M3, andM4) were designed based on theinteractions between AIMP1, RRS, and QRS. The second half(residues 44–72) of the N-terminal helix of AIMP1 were deleted inM1, and the first (residues 7–43) and second halves of the AIMP1helix were exchanged in the M2 mutant. In M3, the first half (resi-dues 1–38) of the N-terminal AIMP1 helix was deleted. In M4, thefirst half of the N-terminal AIMP1 helix (residues 1–30) was re-placed by the leucine-zipper motif (residues 50–80) of AIMP2. TheM1 and M2 mutants were inserted into the pET28a vector, andthe M3 and M4 mutants were inserted into the pCDF vector. To

generate the R1 mutant, five mutations (Lys50Asp, Tyr53Ala,Arg54Asp, Ile57Ala, and Glu65Arg) were made simultaneously inRRS. In the R2 mutant, the Ha helix of RRS (residues 1–35) wasdeleted. The R1 and R2 mutant were inserted into the pET-Duetvector. The AIMP2 leucine-zipper motif (residues 40–100) was in-serted into the pGEX-4T3 vector, expressed in E. coli Rosetta(DE3), and then purified using GST-Sepharose resin with or with-out cleavage of the GST tag by thrombin. The Ha and Hb helices(residues 1–74) of RRS were inserted into the pET28a vector. TheN-terminal helix of AIMP1 (residues 1–73) was inserted into thepCDF vector. RRS-Ha (residues 1–30) and AIMP1 (N-140; resi-dues 1–140) were inserted into thepGEX-4T-3 vector, and RRS-Hb(residues 38–68) with aFLAG tagwas insert into the pET28a vector.

1. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscil-lation mode. Methods Enzymol 276(Pt A):307–326.

2. Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL - a program to evaluate X-ray so-lution scattering of biological macromolecules from atomic coordinates. J Appl Cryst28(Pt 6):768–773.

Sub-complex I

Sub-complex II

AIMP2AIMP2

LRSIRS MRS

QRS QRS

DRS

EPRS

DRS

AIMP1

RRS

AIMP1

EPRS

RRS

AIMP3

Fig. S1. A scheme for the architecture of the MSC complex. The overall architecture of the MSC is shown in a cartoon. In this model, the MSC is divided intothe two subcomplexes. One subcomplex consists of RRS (2), QRS (2), and AIMP1 (2), and the other subcomplex consists of of KRS (4), AIMP2 (2), AIMP3 (1), MRS(1), EPRS (2), IRS (1), LRS (1), and DRS (2). [The numbers in parentheses indicate the number of components in the MSC from rabbit liver based on the reportedbiochemical analysis (1).] We note that the reported stoichiometry of QRS (one QRS) is different from our observations in the present study (two QRS); themodel shown here contains two QRS molecules.

1. Kaminska M, et al. (2009) Dissection of the structural organization of the aminoacyl-tRNA synthetase complex. J Biol Chem 284(10):6053–6060.



A

B D

0

20

40

60

30 40 50 60 70 80 90 100

Abs 28

0

Elu on volume (ml)

440 158 66 29 kDa

E

NN

H1

H2

H3

H4

H5

H6

H7

H8

H9

H11

H12

S1

S2

S3

S4

S5

S8S9

S11

S12

S13

S14

S16

S17

S18

S19

S20

S22S23

S24

S25

Signature motif

(HIGH/VSK)

C

H280

H277

K496S495

Acceptor

binding

domain

Central

domain

Anticodon

binding

domain

V494I278N

NN

Ha

Hb

H1H2

H3

H4

H5

H8H9

H10

H11

H12

H13

H15

H16

H18

H19

C

Signature motif (HVGH/FKT)

S1S2

S3

S5

S6

S7

S9S11

S12

H14

H7T454

K453

H208

H211

Domain I

Domain IIIDomain II

N termimal

domain

F452

V209

N

H208

H211

N202

S200

D197

D388

Y384

Q412

E335

Arg

RRS Active siteY438

D303

E271

F460

R267

T457 P269

R486

L487

K496Gln_AMP

QRS Active site

C

Fig. S2. Overall structures of human RRS and QRS core. (A) Overall structure of RRS in the human RQA1 complex. The N-terminal domain comprises helices Haand Hb. Domain I folds into an α/β structure and binds to the D-loop of tRNAArg. Domain II is a catalytic domain that contains eight α-helices and eight β-strands;this domain binds to Arg, ATP, and the acceptor end of tRNAArg. Domain III comprises seven α-helices and recognizes the anticodon loop of tRNAArg. Residues inthe signature motif of RRS are shown as yellow sticks. The N- and C-terminal ends of RRS are labeled. (B) Close-up view of the RRS active site. The figure is insame orientation as RRS in Fig. 1B, Left). (C) A ribbon diagram of the C-terminal core domain of QRS. The acceptor-binding domain forms an α/β fold and bindsto the acceptor arm of tRNAGln. The central domain contains an active site that binds glutamine and ATP. The anticodon-binding domain binds to the an-ticodon loop of tRNAGln and forms a β-barrel structure with 14 β-strands. The signature motif residues of QRS are shown as yellow sticks. The N- and C-terminalends of QRS are labeled. (D) Close-up view of the QRS active site. The figure is in same orientation as RRS in Fig. 1B, Left. (E) Gel-filtration analysis of the RQA1complex. The molecular weights of the standards are shown above the graph. The weight-average molecular mass of the peak was estimated to be 280 kDa.



H.sapiens RRS 1 M D V L V S E C S A R L L Q Q E E E I - - K S L T A E I D R L K N C G C L - - - - - G A S P N L E Q L Q E E N L K L K Y R L N 56X. laevis RRS 1 M A W H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 4D. melanogaster RRS 1 M S E L N M E L K - K L R E L E L K T - - Q G L A A R I Q T A K S G E Q L - - - - - D - - V D L V Q L Q I E N K K L K N R L F 53C. elegans RRS 1 M S A N K E L Q Q - G Y S D Y S A A S D Q A A L L N R L I T A M Q K G E L T D E L L E H I P E L G D A K K L N D K L K Y R K S 62S.cerevisiae RRS 1 M A S T A N M I S - Q L K K L S I A E - - P A V A K - - - - - - - D S H P - - - - - D - - V N I V - - - - - - - - - - - - - D 33E. coli RRS 1 M N I Q A L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 6

H.sapiens RRS 57 I L R K S L Q A E R N K P T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K N 72X. laevis RRS 5 - F R R V I A E Q V S R V L - - G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L P S - - - - - - - - - - - - E S 23D. melanogaster RRS 54 I L K K S I A E E S T A A G - - G D V S - - - - - - - - - - - - - - - - - - - - - - - - - - K P - - - - - - - - - - - - - K E 75C. elegans RRS 63 I L E K S I A E Q A A K N K K N G V K A T S T S S P S S S T S A P A E K K A K K D G K T G G A P P K Q A K K V D Y V T V E D Y 125S.cerevisiae RRS 34 L M R N Y I S Q E L S K I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 46E. coli RRS 7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 6

H.sapiens RRS 73 M I N I I S R L Q E V F G H A I K A A Y P D L E N P P L L V T P - - S Q Q A K F G D Y Q C N S A M G I S Q M L K T K E - - - Q 130X. laevis RRS 24 C S R V A Q C I Q T V - - - - - - - P V H - - - - - - - - - - - - - - R K H E S P D F Q L - - - - F - M T S L S E G G P N G T 60D. melanogaster RRS 76 S S S I T E H L E S V F R Q A I A S A F P E F R D T P V I I A P V N S T S A K F G D Y Q C N N A M G L S K K L K E K G - - - I 135C. elegans RRS 126 G S S I F G R L Q S L F K K A I E D A F P G L D V - P L L L A E - - T P N P Q F G D Y Q C N S A M P I A A K L K A N K - - - I 182S.cerevisiae RRS 47 - - - - S G V D S S L - - - - - - - I F P A L E W - - - - - - - - - T N T M E R G D L L I - - - - P - I P R L R I K G - - - - 80E. coli RRS 7 - - - - - - - L S E K V R Q A M I A A G A P A D C E P - Q V R Q - - S A K V Q F G D Y Q A N G M M A V A K K L - - - - - - - - 51

H.sapiens RRS 131 K V N P R E I A E N I T K H L P D N - E C I E K V E I A G P G F I N V H L R K D F V S E Q L T S L L - V N G V - - - - - - - Q 184X. laevis RRS 61 F S N Q E E R A E Q L A G K L V C D - S V I S D I G T T R - G S V T F K I N R D L L T R N V L Q Q I S K D G S L Y G L N T E L 121D. melanogaster RRS 136 N K A P R D I A T E L K G H C P A S - P I I E K L E I A G A G F V N V F L S K D Y A S L A L S N L L - R N G V - - - - - - - K 189C. elegans RRS 183 N K R P G D V A K E I Q A K L P T K I D F V E K I D V M P A G F I N I F L N T D Y L R R Q I S L L A - S E G V - - - - - - - K 237S.cerevisiae RRS 81 - A N P K D L A V Q W A E K F P C G - D F L E K V E A N G - P F I Q F F F N P Q F L A K L V I P D I L T R K E - - - - D Y G S 136E. coli RRS 52 G M A P R Q L A E Q V L T H L D L N - G I A N K V E I A G P G F I N I F L D P A F L A E H V Q Q A L A S D R L - - - - - - - G 106

H.sapiens RRS 185 L P A L G E N K K V I V D F S S P N I A K E M H V G H L R S T I I G E S I S R L F E F A G Y D V L R L N H V G D W G T Q F G M 247X. laevis RRS 122 F S E - L S R R K T I V E Y S S P N I A K K F H V G H L R S T I I G N F I A N L K Q A V G N E V V R I N Y L G D W G L Q F G L 183D. melanogaster RRS 190 P P E - V I K K R V L V D F S S P N I A K Q M H V G H L R S T I I G E S L C R L L E F L Q H D V I R I N H L G D W G T Q F G M 251C. elegans RRS 238 L P K - L T R K R V L V D F S S P N I A K E M H V G H L R S T I I G D S I C R L F E A V G F D V L R V N H I G D W G T Q F G M 299S.cerevisiae RRS 137 C K L - V E N K K V I I E F S S P N I A K P F H A G H L R S T I I G G F L A N L Y E K L G W E V I R M N Y L G D W G K Q F G L 198E. coli RRS 107 V A T - P E K Q T I V V D Y S A P N V A K E M H V G H L R S T I I G D A A V R T L E F L G H K V I R A N H V G D W G T Q F G M 168

H.sapiens RRS 248 L I A H L Q D K F P D Y L T V S P P I G D L Q V F Y K E S K K R F D T E - - - - - - - E E F K K R A Y Q C V V L L Q G K N P D 303X. laevis RRS 184 L G A G F S S F G S K E K L L A N P L Q H L F E V Y V K V N T A A E K D - - - - - - - N G I K L S A Q E F L R R L E G G D P H 239D. melanogaster RRS 252 L I A H L E D R F P N Y L N E S P P I S D L Q L F Y K E S K K R F D E D - - - - - - - E E F K K R A Y S R V V S L Q K G V P N 307C. elegans RRS 300 L I A H L Y D R F P D F L K K L P D I S D L Q A F Y K E S K K R F D E D - - - - - - - E Q F K K R A Y E Y V V K L Q S H D G D 355S.cerevisiae RRS 199 L A V G F E R Y G N E E A L V K D P I H H L F D V Y V R I N K D I E E E G D S I P L E Q S T N G K A R E Y F K R M E D G D E E 261E. coli RRS 169 L I A W L E K Q Q Q E N A G - E M E L A D L E G F Y R D A K K H Y D E D - - - - - - - E E F A E R A R N Y V V K L Q S G D E Y 223

H.sapiens RRS 304 I T K A W K L I C D V S R Q E L N K I Y D A L D V S L I - - - E R G E S F Y - Q D R M N D I V K E F E D R - - G F V - Q V D D 359X. laevis RRS 240 A L S L W M H F R D I S I Q E Y A K I Y K R L G A H F D - - D Y S G E S F Y - K E K S Q E V L K Q L E S K - - G L L L K T D K 297D. melanogaster RRS 308 S I K A W E L I C N V S R K E F Q T I Y E R L D I S V K - - - E R G E S F Y - Q S R M L S V V E Y L R G K - - G L L - E V D E 363C. elegans RRS 356 I V K A W N T I C D V S K K Y N Q I V Y N Y L D I K I K - - - D V G E S F Y - Q D K M I E L V K W V K T N K P D M L - R E E D 413S.cerevisiae RRS 262 A L K I W K R F R E F S I E K Y I D T Y A R L N I K Y D - - V Y S G E S Q V S K E S M L K A I D L F K E K - - G L T - H E D K 319E. coli RRS 224 F R E M W R K L V D I T M T Q N Q I T Y D R L N V T L T R D D V M G E S L Y - N P M L P G I V A D L K A K - - G L A - V E S E 282

H.sapiens RRS 360 G R K I V F V P G - - C - - - - S I - - P L T I V K S D G G Y T Y D T S D L A A I K Q R L F E E K A D M I I Y V V D N G Q S V 414X. laevis RRS 298 G T V - I D L S E - - E G N L - S S - - Y S T V M R S D G T S L Y I T R D M A A A V D R M E R Y N F D E M I Y V T D K S Q K N 354D. melanogaster RRS 364 G R E I M W P D D T K T - - - - G I - - P L T I V K S D G G F T Y D T S D M A A I R H R L E E E L C D W I I Y V V D S G Q S T 420C. elegans RRS 414 G R Q I M F P T G - - C - - - - D I - - P L T V V K S D G G F T Y D T S D L A A L K Y R M L E E K C D W N I Y V V D S G Q S L 468S.cerevisiae RRS 320 G A V L I D L T K - - F N K K - L G - - K A I V Q K S D G T T L Y L T R D V G A A M D R Y E K Y H F D K M I Y V I A S Q Q D L 377E. coli RRS 283 G A T V V F L D E - - F K N K E G E P M G V I I Q K K D G G Y L Y T T T D I A C A K Y R Y E T L H A D R V L Y Y I D S R Q H Q 343

Ha Hb

H1 H2

H3 H4

H5

H6 H7 H8

H9 H10

H11

S1 S2

S3 S4

S5 S6

S7 S8

S9 S10 S11

A

Fig. S3. (Continued)



H.sapiens RRS 415 H F Q T I F A A A Q M I G W Y D P K V T R V F H A G F G V V L G E D K K K F K T R S G E T V R L M D L L G E G L K R S M D K L 477X. laevis RRS 355 H F Q Q L F K I L K I M G K - - D Q A D R C Q H V P F G L V Q G - - - - - M K T R R G E V V F L E D V L D E A R S R M L Q N M 410D. melanogaster RRS 421 H F N T I F K A A E R S A I L N P L S H R V D H V Q F G V V L G E D G K K F K T R S G D T V K L S D L L D E G M K R S L Q Q L 483C. elegans RRS 469 H L E T V Y A A G R D F G W Y D E S I Q R V E H V A F G L V L G D D K K K F K T R S G E T V R L L D L L S E G V K R A T E K L 531S.cerevisiae RRS 378 H A A Q F F E I L K Q M G F - - E W A K D L Q H V N F G M V Q G - - - - - M S T R K G T V V F L D N I L E E T K E K M H E V M 433E. coli RRS 344 H L M Q A W A I V R K A G Y V - P E S V P L E H H M F G M M L G K D G K P F K T R S G G T V K L A D L L D E A L E R A R R L V 405

H.sapiens RRS 478 K E K E R D K V L T A E E L N A A Q T S V A Y G C I K Y A D L S H N R L N D Y I F S F D K M L D D R G N T A A Y L L Y A F T R 540X. laevis RRS 411 A A S E T S K - - Q L D D P S D T A E K I G I A A L I V Q D F K G Q L V S D Y H F D W D Q A L Q S T G D T G V F L Q Y T H A R 471D. melanogaster RRS 484 E S R G R D K V L T P Q E L K D A Q E S L A Y G C I K Y S D L C H N R I S D Y I F S F D K M L E D R G N T A V Y L L Y T Y T R 546C. elegans RRS 532 I E K G R E T A M S E E Q L V A A R D A V A F G C V K Y A D L S H T R T Q D Y V F S F D R M L E D R G N T A V Y L L Y A Y T R 594S.cerevisiae RRS 434 - K K N E N K Y A Q I E H P E E V A D L V G I S A V M I Q D M Q G K R I N N Y E F K W E R M L S F E G D T G P Y L Q Y A H S R 495E. coli RRS 406 A E K N P D - - M P A D K L E T L A N A V G I G A V K Y A D L S K N R T T D Y I F D W D N M L A F E G N T A P Y M Q Y A Y T R 466

H.sapiens RRS 541 I R S I A R L A N - I D E E M - L Q K A A R E T K I L L D H E K E W K L G R C I L R F P E I L Q K I L D D L F L H T L C D Y I 601X. laevis RRS 472 L H S L Q A L R C P R D T E D - F - - - E I A - - - S L R E P C V I A T L Q H L L R Y D E V I Y K C L E D L Q P R Y L V T Y L 527D. melanogaster RRS 547 I C S I A R N S G - E D F T N - L P E I L K K T N I V L D H E K E W K L A K T L L K L H D I L I K C S K E L F L H F L C E F C 607C. elegans RRS 595 I Q S I F E K D E - V K N V D L V K Y I A S T P T L P L D H P G E F K L A K Q L L K L S D C V L L V L D S L M L H Q M C D Y V 656S.cerevisiae RRS 496 L R S V E R N A S G I T Q E K - W - - - I N A D F S L L K E P A A K L L I R L L G Q Y P D V L R N A I K T H E P T T V V T Y L 554E. coli RRS 467 V L S V F R K A E - I N E E Q - L - - - A A A - P V I I R E D R E A Q L A A R L L Q F E E T L T V V A R E G T P H V M C A Y L 523

H.sapiens RRS 602 Y E L A T A F T E F Y D S C Y C V E K D R Q T G K I L K V N M W R M L L C E A V A A V M A K G F D I L G I K P V Q R M 660X. laevis RRS 528 L S L G H L A N V A H R T L Q V - K - - - - - G S S S E A A H A R L L F F K N V Q M V L G N G M K L L G I T P V N N M 580D. melanogaster RRS 608 F E V C T V F T E F Y D S C Y C I E K N - K Q G D I I G V N H S R I L L C E A T A A V L R Q C F Y I L G L K P V S K M 665C. elegans RRS 657 Y Q L A T L F H D F Y N E C Y V I E N - - K E G E K P F V H M H R L A L C D V T R K V M S T C F K I L G L R E V N K M 713S.cerevisiae RRS 555 F K L T H Q V S S C Y D V L W V - A - - - - - G Q T E E L A T A R L A L Y G A A R Q V L Y N G M R L L G L T P V E R M 607E. coli RRS 524 Y D L A G L F S G F Y E H C P I L S - - - - - A E N E E V R N S R L K L A Q L T A K T L K L G L D T L G I E T V E R M 577

Signature motifActive sitep43/AIMP1 interaction

H12 H13

H14 H15

H16 H17 H18

H29

S12

QRS interaction

HighRowconserved conserved




H.sapiens QRS 1 M A A L D S L - S L F T S L G L S E Q K A R E T L K N S A L S A Q L R E A A T Q A Q Q T L G S T I D K A T G I L L Y G L A S R 62X. laevis QRS 1 M A A E - S L - S L F T A I G L S E Q K A R E T L K N E A L S L L L R D A I V Q A Q K T L G P S V D K V T G T L L Y N V A T R 61D. melanogaster QRS 1 M A G D D L I - A K F Q A L G M S E Q K A K E T L K N A N V T K N L Q L S L A A A - G - - S A T L S D G T G M L I Y H M A T K 59C. elegans QRS 1 M A T K E E L - - - - L S L G L S D S K V A E T L K N V K L T E T I G S I V K L A S E - - S G E I S K Q K G T L L Y Q L A T K 57S.cerevisiae QRS 1 M S S V E E L T Q L F S Q V G F E D K K V K E I V K N K K V S D S L Y K L I K E T P S - - D Y Q W N K S T R A L V H N L A S F 61E. coli QRS 1 M - - - - - - - - - - - - - - - S E A E A R P T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 10

H.sapiens QRS 63 L R D T - - R R L S F L V S Y I A S K K I H T E P Q L S A A L E Y V R S H P L - - - D P I D T V D F E R E C G V G V I V T P E 120X. laevis QRS 62 L K D T - - K R L G F L V E Y I A S K K I T T D L Q L N A A L D Y V K A H P L - - - D P I D T G H F E K D C G V G V T V T P E 119D. melanogaster QRS 60 L K P Q T A D H L P L L V R Y I V E H K L D N T Q R V D A A L E Y L L K C G Q S L N A N I D L Q A L E K E C G V G V V V T P E 122C. elegans QRS 58 L K P Q V A A H T P L V V K Y I M N D G I K T E P Q L S A A I E Y L L S H T V - - - K G I Q V P D F E K S C G V G V V V T I D 117S.cerevisiae QRS 62 V K G T D L P K S E L I V N G I I N G D L K T S L Q V D A A F K Y V K A N G E - - - A S T K M - G M N E N S G V G I E I T E D 120E. coli QRS 11 - - - - - - - - - - - F I R Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 14

H.sapiens QRS 121 Q I E E A V E A A I N - R H R P Q L L V E R Y H F N M G L L M G E A R - - A V L K W A D G K M I K N E V D M Q V L H L L G P K 180X. laevis QRS 120 Q I E E A V E A V I Q - K F R A Q L L A E R Y R F N M G L L M G E A R - - N Q L K W A D G K I I K N E V D M Q V L H L L G P K 179D. melanogaster QRS 123 Q I E R T V Q A K I K A S Y K E A L L E Q R Y H F N S F K I L Q D V R - - G E L K W A D A K S V K A A I D V E I F D L L G P K 183C. elegans QRS 118 D I E A A V T K V I G - Q H R E K I V A E R Y S F P A G K L L G E L R - - A L L P W A D G A I T K K E V D L R F L E L L G P K 177S.cerevisiae QRS 121 Q V R N Y V M Q Y I Q - E N K E R I L T E R Y K L V P G I F - A D V K N L K E L K W A D P R S F K P I I D Q E V L K L L G P K 181E. coli QRS 15 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 14

H.sapiens QRS 181 L E A D L E K K - F K V A - K A R L E E T D R R T A K D V V E N - G - - - - - - - - - E T A D Q T L S L M E Q L R G E A L K F 231X. laevis QRS 180 T E A D L E K K - P K V T - K P K A T E P E K K - - G D A T V N - G - - - - - - - - - D V K L E S V S L M E Q L R G E A L K F 228D. melanogaster QRS 184 T E A D L K P Q - T K A N D K P K A A K P K A E V - T P A A Q T - A - - - - - - - - - E A A S D G A T T I S E L M K T K V H F 234C. elegans QRS 178 T A E D L A P K - K K E K - K P E G P K P S K D A - A A A A T A P G T K N Q K E A S P E E F A D G A E T M D E L L R T R A H F 237S.cerevisiae QRS 182 D E R D L I K K K T K N N - E K K K T N S A K K S - S D N S - - - - - - - - - - - - - A S S G P K R T M F N E - - G F L G D L 227E. coli QRS 15 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 14

H.sapiens QRS 232 H K P G E N Y K T P G Y V V T P H T M N L L K Q H L E I - T G G Q V R T R F P P E P N G I L H I G H A K A I N F N F G Y A K A 293X. laevis QRS 229 H K P G E N Y K T E G Y V V T P K T M E L L K K H L E I - T G G Q I R T R F P P E P N G I L H I G H A K A I N F N F G Y A K A 290D. melanogaster QRS 235 H A P G E N F K A D G Y V V T E H T E R L L K E H L A R - T G G K V H T R F P P E P N G I L H I G H A K A I N I N F G Y A A A 296C. elegans QRS 238 H K V G E N F K Q D G Y V T T P K T A E L L K A H V A A - V G G K V V T R F P P E P N G V L H I G H A K A I N I N F G Y A K A 299S.cerevisiae QRS 228 H K V G E N P Q A - - - - - - - - Y P E L M K E H L E V - T G G K V R T R F P P E P N G Y L H I G H S K A I M V N F G Y A K Y 281E. coli QRS 15 - - - - - - - - - - - - - - - - - - - - I I D E D L A S G K H T T V H T R F P P E P N G Y L H I G H A K S I C L N F G I A Q D 57

H.sapiens QRS 294 N N G I C F L R F D D T N P E K E E A K F F T A I C D M V A W L G Y T - P Y K V T Y A S D Y F D Q L Y A W A V E L I R R G L A 355X. laevis QRS 291 N G G I C F L R Y D D T N P E K E E E K Y F T A I K D M V E W L G Y K - P Y A V T H A S D N F N Q L Y E W A V E L I R R G Q A 352D. melanogaster QRS 297 H D G V C Y L R Y D D T N P E K E E E K F F L A I K E M V E W L G Y K - P F K I T Y S S D N F Q Q L Y E W A V V L I N K G L A 358C. elegans QRS 300 M G G V C N L R F D D T N P E K E E E K F F S A I E D I V H W L G Y D - P A R V T H S S D N F Q Q L Y L W A V K L I Q K G L A 361S.cerevisiae QRS 282 H N G T C Y L R F D D T N P E K E A P E Y F E S I K R M V S W L G F K - P W K I T Y S S D Y F D E L Y R L A E V L I K N G K A 343E. coli QRS 58 Y K G Q C N L R F D D T N P V K E D I E Y V E S I K N D V E W L G F H W S G N V R Y S S D Y F D Q L H A Y A I E L I N K G L A 120

H.sapiens QRS 356 Y V C H Q R G E E L K G H N T - - - - - - - - - - - L P S P W R D R P M E E S L L L F E A M R K G K F S E G E A T L R M K L V 407X. laevis QRS 353 Y V C H Q K V E E I K G H N P - - - - - - - - - - - P P S P W R D R P V E E S L L L F E G M K K G K F A E G E A T L R M K L I 404D. melanogaster QRS 359 Y V C H Q K A E E L K G F N P - - - - - - - - - - - K P S P W R E R P I E E S L R L F E D M K R G K I D E G A A T L R M K V T 410C. elegans QRS 362 F V C H Q K V E E M R G F E V - - - - - - - - - - - Q L S P W R E R P I E E N I Q L F E D M K N G K F D E G E A T L R L K L T 413S.cerevisiae QRS 344 Y V C H C T A E E I K R G R G I K E D G T P G G E R Y A C K H R D Q S I E Q N L Q E F R D M R D G K Y K P G E A I L R M K Q D 406E. coli QRS 121 Y V D E L T P E Q I R E Y R G T L T Q P - - - - - G K N S P Y R D R S V E E N L A L F E K M R A G G F E E G K A C L R A K I D 178

H.sapiens QRS 408 M E D G - - - K M D P V A Y R V K Y T P H H R T G D K W C I Y P T Y D Y T H C L C D S I E H I T H S L C T K E F Q A R R S S Y 467X. laevis QRS 405 M E D G - - - K M D P V A Y R I K Y T P H H R T G D K W C I Y P T Y D Y T H C L C D S I E H I T H S L C T K E F Q A R R S S Y 464D. melanogaster QRS 411 L E E G - - - K M D P V A Y R I K F I S H H R T G S D W C I Y P T Y D Y T H C L C D S L E D I T H S L C T K E F Q S R R S S Y 470C. elegans QRS 414 L E E G - - - K V D P V A Y R I K Y V P H H R T G N Q W C I Y P T Y D Y T H C L C D S I E N I T H S L C T K E F Q S R R S S Y 473S.cerevisiae QRS 407 L N S P S P Q M W D L I A Y R V L N A P H P R T G T K W R I Y P T Y D F T H C L V D S M E N I T H S L C T T E F Y L S R E S Y 469E. coli QRS 179 M A S P F I V M R D P V L Y R I K F A E H H Q T G N K W C I Y P M Y D F T H C I S D A L E G I T H S L C T L E F Q D N R R L Y 241

H1

H2 H3

H4 H5

H6

H7

S1

S2 S3

S4 S5

S6 S7 S8

B




H.sapiens QRS 468 F W L C N A L D V - Y C P V Q W E Y G R L N L H Y A V V S K R K I L Q L V A T G A V R D W D D P R L F T L T A L R R R G F P P 529X. laevis QRS 465 F W L C N T L D V - Y C P V Q W E Y G R L N L H Y T V V S K R K I I K L V E T G A V R D W D D P R L F T L T A L R R R G F P P 526D. melanogaster QRS 471 Y W L C N A L G I - Y C P V Q W E Y G R L N M N Y A L V S K R K I A K L I T E Q I V H D W D D P R L F T L T A L R R R G F P A 532C. elegans QRS 474 Y W L C N A L D I - Y C P V Q W E Y G R L N V N Y T V V S K R K I L K L I T T K T V N D W D D P R L F T L T A L R R R G I P S 535S.cerevisiae QRS 470 E W L C D Q V H V - F R P A Q R E Y G R L N I T G T V L S K R K I A Q L V D E K F V R G W D D P R L F T L E A I R R R G V P P 531E. coli QRS 242 D W V L D N I T I P V H P R Q Y E F S R L N L E Y T V M S K R K L N L L V T D K H V E G W D D P R M P T I S G L R R R G Y T A 304

H.sapiens QRS 530 E A I N N F C A R V G V T V A Q T T M E P H L L E A C V R D V L N D T A P R A M A V L E S L R V I I T N F P A A - K S L D I Q 591X. laevis QRS 527 E A I N N F C A R V G V T V A Q T T M E P H L L E S C V R D V L N E T A P R I M A V L E P L K V T I T N L P A E - K A I D V S 588D. melanogaster QRS 533 E A I N N F C A Q M G V T G A Q I A V D P A M L E A A V R D V L N V T A P R R L V V L E P L K V T I K N F P H A - A P V Q L E 594C. elegans QRS 536 E A I N R F V A K L G L T M S Q M V I D P H V L D A T V R D Y L N I H A P R T M A V L E G L K L T I E N F S E L N L P S S V D 598S.cerevisiae QRS 532 G A I L S F I N T L G V T T S T T N I Q V V R F E S A V R K Y L E D T T P R L M F V L D P V E V V V D N L S D D - Y E E L A T 593E. coli QRS 305 A S I R E F C K R I G V T K Q D N T I E M A S L E S C I R E D L N E N A P R A M A V I D P V K L V I E N Y Q G E - G - E M V T 365

H.sapiens QRS 592 V P N F P A D E T K G - F H Q V P F A P I V F I E R T D F K E - E - P E P G F K R L A W G Q P V G L R H T G Y V I E L Q H V V 651X. laevis QRS 589 V P N F P A D E S K G - F H V V P F S S T I Y I E Q S D F R E - V - M E K G Y K R L T P D Q P V G L R H A G Y V I S L Q N I V 648D. melanogaster QRS 595 V P D F P Q N P Q Q G - T H K I T L D K V I Y I E Q G D F K L - E - P E K G Y R R L A P K Q S V G L R H A G L V I S V D E I V 654C. elegans QRS 599 V P D F P S D P T D P R K H S V S V D R E I F I E K S D Y K P D D - S D K S F R R L T P K Q A V G L K H I G L V L R F V K E V 660S.cerevisiae QRS 594 I P Y R P G T P E F G - E R T V P F T N K F Y I E R S D F S E - N V D D K E F F R L T P N Q P V G L I K V S H T V S F K S L E 654E. coli QRS 366 M P N H P N K P E M G - S R Q V P F S G E I W I D R A D F R E - E - A N K Q Y K R L V L G K E V R L R N A - Y V I K A E R V E 424

H.sapiens QRS 652 K G P - S G C V E S L E V T C R R - - - - - - A D A G E K P K A F I H W V S Q - - - - - - P L M - C E V R L Y E R L F Q H K N 700X. laevis QRS 649 K D Q - S G N V I E L E V T C T K - - - - - - T D V A E K P K A F I H W V S D - - - - - - P L T - C E V R L Y D R L F L H K T 697D. melanogaster QRS 655 K D P A T G Q V V E L I C T S Q P - - - - - - A E Q A E K P K A F V Q W V S Q - - - - - - P I Q - L E V R L Y E Q L F K H K N 704C. elegans QRS 661 K D A - E G H V T E V V V K A E K - - - - - - L S E K D K P K A F I H W V A K - - - - - - P V S - C E V R L Y D R L F K S K N 709S.cerevisiae QRS 655 K D E - A G K I I R I H V N Y D N - - K V E E G S K P K K P K T Y I Q W V P I S S K Y N S P L R V T E T R V Y N Q L F K S E N 714E. coli QRS 425 K D A - E G N I T T I F C T Y D A D T L S K D P A D G R K V K G V I H W V S A A - - - - H A L P - V E I R L Y D R L F S V P N 481

H.sapiens QRS 701 P E D P T E V P G G F L S D L N L A S L H V V D A A L V D C S V - - - - - - - - - - - - - - - - - - - - - - - - A L A K P F D 739X. laevis QRS 698 P E D P S E V P G G F L T D L N T N S L T T I P S A L V E R S V - - - - - - - - - - - - - - - - - - - - - - - - K N A K A L D 736D. melanogaster QRS 705 P E D P N E V P G G F L S D I S E Q S M S V V - V A F A D R A L - - - - - - - - - - - - - - - - - - - - - - - - N Q A K V Y D 742C. elegans QRS 710 P E D A Q L V P G G F L S D I N P D S L T V V Y N A L I D Q S I - - - - - - - - - - - - - - - - - - - - - - - - A K S K V Y D 748S.cerevisiae QRS 715 P S S - - - H P E G F L K D I N P E S E V V Y K E S V M E H N F G D V V K N S P W V V D S V K N S E F Y V E E D K D S K E V C 774E. coli QRS 482 P G A - - - - A D D F L S V I N P E S L V I - K Q G F A E P S L - - - - - - - - - - - - - - - - - - - - - - - - K D A V A G K 515

H.sapiens QRS 740 K F Q F E R L G Y F S V D P - D S H Q G K L V F N R T V T L K E D - - P G - - K V 775X. laevis QRS 737 K F Q F E R L G Y F S V D P - D T T P E K I V F N R T V T L K E D - - P G - - K M 772D. melanogaster QRS 743 K F Q F E R I G F F S V D P - D T S A N H L V F N R T V G L K E D - - A G - - K K 778C. elegans QRS 749 R F Q F E R I G F F C V D R - D S T S S T L V F N R T V M L K D G G A S G - - K N 786S.cerevisiae QRS 775 R F Q A M R V G Y F T L D K - E S T T S K V I L N R I V S L K D A - - - T - - S K 809E. coli QRS 516 A F Q F E R E G Y F C L D S R H S T A E K P V F N R T V G L R D T - - W A K V G E 554

β25β23 β24

H8 H9 H10

H11 H12

S9 S10

S11 S12 S13

S14 S15 S16 S17 S18

S19 S20 S21

S22

Signature motifActive sitep43/AIMP1 interactionRRS interaction

HighRowconserved conserved

Fig. S3. Structure-based sequence alignment of RRS and QRS homologs. (A) The secondary structure of human RRS is displayed above the sequences. Thelocations and functions of the key residues are highlighted and annotated. H, α-helix, S, β-sheet. (B) The secondary structure of human QRS is displayed abovethe sequences. The locations and functions of the key residues are highlighted and annotated.



Flexible loop

Fig. S4. Model of the conformational change in RRS in the hexameric subcomplex structure that allows tRNAArg binding. The mobile loop (black arrow)between the Ha and Hb helices of RRS may allow rigid body movement of the RRS core and AIMP1 helix, which may result in the opening of the active site ofRRS. The active sites are marked with stars.



BA

0.01 0.11E-6

1E-4

0.01

1

M4

M3

log

I(a.

u.)

q (Å-1)

WT

0.5

0 50 100 150 200 2500.000

0.004

0.008

0.012

p(r)

(a.u

.)

r (Å)

WTM3M4

C D

E

0

20

40

60

80

50 55 60 65 70 75 80 85 90

Abs

280

Elu on volume (ml)

WT

M3

M4

440 158 66 kDa

WTQRSRRS

A1_WT

M4

M3

QRS

RRSA1_M3

QRSRRSA1_M4

ml 62 65 67 71 73

ml 64 65 68 71 74

ml 61 63 65 67 70

0

20

40

60

80

100

50 55 60 65 70 75 80 85 90

Abs

280

Elu on volume (ml)

WT

M3

M4

440 158 66 kDa QRS

RRS

A1_WT

QRS

RRS

A1_M3

QRS

RRS

A1_M4

KRS

A2

KRS

A2

KRS

A2

WT

M4

M3

ml 62 64 65 69 74

ml 61 63 66 68 71

ml 60 63 66 68 71

0

50

100

150

200

250

300

350

50 55 60 65 70 75 80 85 90

Abs

280

Elu on volume (ml)

R2_WT

R2_M3

R2_M4

440 158 66 kDa

QRS

RRS_R2A1_M3

R2_M3

QRS

RRS_R2A1_WT

R2_WT

QRS

RRS_R2A1_M4

R2_M4

ml 59 61 63 66 68

ml 66 68 71 73 75

ml 67 69 72 74 76

F

Fig. S5. Molecular assembly of the RQA1 or higher-order ARS subcomplex. (A) The schematic diagram of AIMP1 (M1, M2, M3, and M4) and RRS (R1 and R2)mutants used in this study. (B) Gel-filtration analyses of wild-type RQA1 (red), M3 mutant RQA1 (orange), and M4 mutant RQA1 (blue). The molecular weightsof the standards are shown above the graphs. The elution volume of each lane is labeled on the top of gel in the color used in the graph. (C) Gel-filtrationanalyses of wild-type RQA1KA2 (red), M3 mutant RQA1KA2 (orange), and M4 mutant RQA1KA2 (blue). (D) The SAXS profile of the solution structure (1 mg/mL)of the RQA1 subcomplex containing wild-type AIMP1 (squares), the M3 (circles), or the M4 (triangles) mutant. The solid red (wild-type AIMP1), orange (M3mutant), and blue (M4 mutant) lines are the theoretical SAXS curves obtained from the reconstructed structures of the hexameric forms of human ARS withlowest χ value using the ab initio shape-determination program DAMMIF (1). For clarity, each plot is shifted along the log I axis. (E) Pair distance distributionfunction p(r) for the RQA1 complex containing wild-type AIMP1 (red) or the M3 (orange) or M4 (blue) mutant based on an analysis of the experimental SAXSdata. (F) Gel-filtration analyses of the ternary complex containing the RRS R2 mutant, QRS, and wild-type AIMP1 (green) or the M3 (purple) or M4 (red) mutant.

1. Franke D, Svergun DI (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Cryst 42(Pt 2):342–346.



input

FLAG_RRS_Hb

FLAG_RRS_Hb

WB: GST

RRS_Ha AIMP1_N140GST_tagged:

FLAG_RRS_Hb:

AIMP1_N140

RRS_HaGST

WB: FLAG

A

B

-80

-60

-40

-20

0

20

40

60

80

100

190 200 210 220 230 240 250

Mea

n re

sidu

al m

olar

elli

ptic

ity(d

eg∙c

m2

dmol

-110

-3)

Wavelength (nm)

RRS_N74

Fig. S6. The structural and biochemical features of the N-terminal helices of RRS. (A) Circular dichroism spectra of the N-terminal domain of RRS (N-74) showedthat the region forms the α-helix structure. Structure of the RRS (N-74) fragment was monitored by CD spectrophotometer (Jasco J-715) at wavelengths of190–250 nm at 18 °C. (B) Western blot of the GST-RRS (Ha) and FLAG-RRS (Hb). Purified GST-tagged protein was mixed with FLAG-RRS (Hb) at a 1:1 molar ratioand was incubated at 4 °C for 3 h in the presence of glutathione resin. Blots were probed with antibody (α-GST or α-FLAG) directed against GST (control),GST-RRS (Ha), GST-AIMP1 (N-140), and FLAG-RRS (Hb).



AIMP1 RRS_Hb

g

a

eb

f

c

d

d

a

eb

f

cg

A69L62L55L48

R49K56K63E70

V50K57Q64I71

Q72E65E58E51

A53E60I67G74

N52I59L66N73

V75Q68E61K54

L44L51L58E65

R66R59K52Q45

E46Y53K60N67

N40E47R54S61

L62L55N48L41

Q63N56L49E42

Q43K50I57A64

C8Q15L22L29

I26I19L12V5

L32L25A18L11

L8G15I22V29

AIMP1 RRS_Ha

g

a

eb

f

c

d

d

a

eb

f

cg

E12D19K26K33

Q13Q20Q27E34

K35Q28I21K14

K9A16E23S30

L31Y24E17R10

T23E16S9D2

V3A10E17A24

L4R11E18E25

D27K20L13S6

E7Q14S21R28

A

B

Fig. S7. Helical wheel diagram of the coiled-coil interactions between the N-terminal helices of AIMP1 and RRS. (A) The parallel coiled-coil interactionsbetween the Hb helix of RRS and the second half of the AIMP1 helix. (B) The coiled-coil interface between the Ha helix of the symmetry-related RRS proteinand the first half of the AIMP1 helix. The two helices are directed in a parallel manner. The leucine-zipper interactions are highlighted in yellow.



Table S1. Data collection and refinement statistics for RRS-QRS-AIMP1

Data collectionSpace group C2221Cell dimensions

a, b, c, Å 102.7, 313.3, 161.8α, β, γ, ° 90, 90, 90

Resolution, Å* 50–3.85(3.92–3.85)Rsym 11.5(79.9)I /σI 22.3(2.9)Completeness (%) 99.5(99.8)Redundancy 5.1(5.0)

RefinementResolution, Å 37.9–4.0No. reflections 15,364Rwork/Rfree, % 23.7/28.7No. atoms†

Protein 10,246B-factors, Å2

RQA1 155.1RRS 166.9QRS 140.1AIMP1 159.1

RmsdBond lengths, Å 0.004Bond angles, ° 0.874

*Values in parentheses are for highest-resolution shell.†The final model consists of residues 2–660 of RRS, residues 220–771 of QRS,and residues 5–80 of AIMP1 with 95.1% favored and 4.9% outliers inRamachandran plot. The N terminus of QRS and C terminus of AIMP1 werenot visible.

Table S2. Structural parameters obtained from the SAXS dataof the MSC subcomplex in solution

Sample Rg,G, Å* Rg,p(r), Å† Dmax, Å

†

RQA1 subcomplexRQA1 trimeric crystal 49.00 ± 0.05 49.06 ± 0.04 167.8RQA1 hexameric crystal 56.20 ± 0.01 56.26 ± 0.04 187.6RQA1_WT 56.40 ± 0.49 57.91 ± 0.59 163.5RQA1_M3 67.09 ± 0.96 67.14 ± 1.38 208.5RQA1_M4 58.52 ± 0.54 58.60 ± 0.26 168.9

RQA1-KA2 subcomplexRQA1-KA2 77.10 ± 1.48 80.48 ± 0.22 258.4

*Radius of gyration obtained from the scattering data by the Guinier analysis.†Rg,p(r) (radius of gyration) and Dmax (maximum dimension) were calculatedfrom the p(r) function by the program GNOM (1).

1. Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 25(Pt 4):495–503.



Table S3. Kinetic parameters for tRNA of RRS and QRS in the MSC subcomplex

Enzyme Human tRNAArg or tRNAGln Km, mM kcat/s kcat/KM·s−1·mM−1

RRS/QRSFull length RRS 4.529 ± 0.187 1.115 ± 0.059 0.246Full length QRS 4.021 ± 0.112 1.27 ± 0.087 0.316

RQA1 subcomplexR-Q-A1(WT) RRS 4.826 ± 0.155 1.121 ± 0.078 0.232

QRS 3.320 ± 0.091 1.15 ± 0.021 0.346R-Q-A1(M3) RRS N.D. N.D. N.D.

QRS 3.126 ± 0.035 1.031 ± 0.008 0.330R-Q-A1(M4) RRS N.D. N.D. N.D.

QRS 3.408 ± 0.038 1.09 ± 0.011 0.320R(R2)-Q-A1(WT) RRS 4.206 ± 0.053 1.125 ± 0.017 0.267

QRS 3.156 ± 0.037 0.958 ± 0.016 0.304R(R2)-Q-A1(M3) RRS 4.044 ± 0.073 1.068 ± 0.058 0.264

QRS 3.956 ± 0.041 1.163 ± 0.007 0.294R(R2)-Q-A1(M4) RRS 4.018 ± 0.097 1.077 ± 0.098 0.268

QRS 3.325 ± 0.032 0.923 ± 0.018 0.278RQA1–KA2 subcomplexR-Q-A1-K-A2(WT) RRS 5.370 ± 0.057 1.201 ± 0.021 0.224

QRS 7.674 ± 0.070 2.23 ± 0.013 0.291R-Q-A1(M3)-K-A2 RRS N.D. N.D. N.D.

QRS 7.938 ± 0.159 1.807 ± 0.026 0.228R-Q-A1(M4)-K-A2 RRS N.D. N.D. N.D.

QRS 6.912 ± 0.132 1.512 ± 0.063 0.219

N.D., not detected.



supporting information - pnas · 2014. 10. 5. · supporting information fu et al....

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